Purified and modified hemoglobins are potential blood substitutes, however, significant toxicities are associated with their in vivo application. We have studied the effects of purified hemoglobin A0 (HbA0) (10-9 - 10-6 M) on platelet reactivity. HbA0 did not activate platelets when added alone but potentiated agonist-induced platelet aggregation in a concentration dependent manner. HbA0 (10-6 M) nearly doubled the extent of platelet aggregation induced with submaximal concentrations of collagen, thrombin and arachidonic acid but did not potentiate aggregation induced with ADP or ADP/epinephrine. The potentiation was not blocked by indomethacin, apyrase, yohimbine, superoxide dismutase or catalase. Heme-mediated reactions only partially accounted for this potentiation since methemoglobin A0, carbon monoxide- treated HbA0 and cyanomethemoglobin A0 potentiated aggregation 62-97% as well as native hemoglobin A0. Affinity chromatography of solubilized platelet membranes on hemoglobin-agarose revealed an interaction of hemoglobin with a 125 kDa and a 95 kDa protein. These proteins were identified as the aIIbBeta3 integrin complex (glycoprotein IIb/IIIa) by immunoblotting with monoclonal antibodies. This interaction was prevented with free HbA0 in solution suggesting a specific interaction. Hemoglobin may potentiate platelet aggregation by modulating the interaction of integrin aIIbBeta3 and fibrinogen.The significance of this project lies in identifying interactions between circulating blood cells and hemoglobin-based blood substitutes in vitro and thus predict potential toxic reactions when the blood substitutes are used in vivo.